HIV-1 Env protein structure and function assessed by parallel smFRET and cryoET Summary The HIV-1 Env protein mediates virus entry into cells by promoting fusion between viral and cellular membranes. As the only virus protein on the surface of virus particles, HIV-1 Env is also a major target for vaccines. The greatest barriers to the development of a vaccine have been the high sequence variability of Env, its dense glycan shield and conformational dynamics. We have applied smFRET imaging to gain insights into the conformational states and conformational dynamics of individual Env protomers in the context of the native trimer on the surface of intact virions. Our work has revealed that Env opens from a pre-triggered conformation (State 1) through a necessary intermediate (State 2), into a three CD4 receptor-bound conformation (State 3). Many broadly neutralizing antibodies (bNAbs) generated by few patients were found to exhibit a preference for State 1. Associating these conformational states with existing high-resolution structures, we made the surprising observation that the constructs upon which extant high-resolution structures are based predominantly occupy the downstream State 2 conformation. Hence, the high-resolution structure of the pre-triggered State 1 Env, the predominant conformational state of Env on the surface of viruses, which is preferentially recognized by most broadly neutralizing antibodies (bNAbs) - and thus of central vaccine importance - remains unknown. To validate and explore these unexpected findings we propose to combine smFRET with other structural methods to gain a comprehensive understanding of the structure and dynamics of the native trimer on the surface of intact virions. Towards this end we have established closely knit collaborations between our existing Mothes/Blanchard team and the laboratory of Dr. Jun Liu to determine the structure of the HIV-1 Env trimer in its distinct states, including State 1, on the surface of virions by cryo- electron tomography (cryoET). Leveraging the inherent synergies of smFRET and cryoET, we will capture and determine structural intermediates of viral entry during membrane fusion.